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Glyceraldehyde 3-phosphate dehydrogenases
J. Mol. Biol. (1965) 13, 876-884
Glyceraldehyde 3-Phosphate Dehydrogenases I. The Protein Chains in Glyceraldehyde 3-Phosphate Dehydrogenase from Pig Muscle J.
lEUAN HARRIS AND
R. N.
PERHAM
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 4 June 1965) Glyceraldehyde 3-phosphate dehydrogenases have been studied by methods of structural protein chemistry. Derivatives of the pig muscle enzyme prepared by oxidation of the native enzyme with performic acid, and by carboxymethylation with iodo[I-14C]acetic acid in 8 M-urea, have been shown to contain four unique residues of cysteic acid, and of S-[l-14C]carboxymethylcysteine, respectively, indicating that the structural monomer in the enzyme contains four unique cysteines and that disulphide bridges do not contribute to its molecular structure. Amino-acid and end-group analysis, and a study of tryptic digests by peptide mapping techniques have shown further that the protein monomer in the pig muscle enzyme consists of a single polypeptide chain containing approximately 330 amino acid residues, corresponding to a molecular weight of 36,000 ± 1000. It is proposed that the active enzyme comprises four such identical protein chains, each of which contains one reactive cysteine and combines with one molecule of coenzyme (NAD), to form jour structurally independent catalytic sites within the quaternary structure of the active tetramer.
1. Introduction Glyceraldehyde 3-phosphate dehydrogenase (GPDH, E.C. no. 1. 2 .1.12) is a key enzyme in the glycolytic conversion of glucose to pyruvic acid and as such has an important role in the carbohydrate metabolism of most organisms (for review see Velick & Furfine, 1963). The enzyme was first obtained in pure crystalline form from yeast (Warburg & Christian, 1939) and from rabbit skeletal muscle (Caputto & Dixon, 1945; Cori, Slein & Cori, 1945). It has since been isolated from a variety of other sources, notably by Elodi & Szorenyi (1956) and Allison & Kaplan (1964). The enzyme from rabbit muscle has been variously reported to possess a molecular weight of 118,000 (Elias, Garbe & Lamprecht, 1960), 120,000 (Taylor & Lowry, 1956), 140,000 (Dandliker & Fox, 1955; Fox & Dandliker, 1956) and 142,000 (EMdi, 1958), and this apparent variability in molecular weight is reflected in the values reported for other parameters of the active enzyme, such as the number of moles of bound coenzyme (NAD) and the number of catalytically active sites. A molecular weight of 120,000 has been frequently adopted; on this basis the rabbit enzyme was thought to combine with 3 moles of NAD, and to contain from 11 to 14 titratable SH groups, of which 3 appeared to be highly reactive and essential for activity (cf. Velick & Furfine, 1963). 876
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
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The determination of the amino acid sequences around the reactive cysteines in GPDH'st following their reaction with the inhibitor, iodo[l- 14C]acetic acid, and the substrate, p-nitrophenyl[l- 14C]acetate, respectively, showed that these particular cysteines occurred in the same octadecapeptide sequence in the primary structure of the enzyme protein. These results led us to suggest that a molecule of active enzyme contained at least three catalytic sites and that it was composed of an equal number of similar, if not identical, protein chains (Harris, Meriwether & Park, 1963; Harris & Perham, 1963). At the same time it was apparent that this estimate of the number of constituent protein chains represented a minimum value, which was based on the assumption that the enzyme preparations used for these studies were composed wholly of fully active enzyme molecules. If, on the other hand, they contained a proportion of protein chains which were not capable of reacting with the inhibitor or the substrate, then the above estimate of the number of protein chains comprising the enzyme molecule could well be too low. In an endeavour to eliminate this possible source of error, we have sought to establish the number, and molecular size, of the protein chains in glyceraldehyde 3-phosphate dehydrogenases by methods which do not necessarily depend on the availability offully active enzyme. A study of the enzyme protein, involving amino-acid and endgroup analysis and the characterization of polypeptide fragments produced by trypsin digestion of the carboxymethylated derivatives of rabbit and pig muscle GPDH's, has now revealed that the structural monomer in both enzymes appears to consist of a protein chain containing approximately 330 amino acids, corresponding to a chain molecular weight of 35,000 to 36,000 (Harris & Perham, 1963,1964). On this basis, active enzyme consisting of three of these protein chains would be expected to possess a molecular weight of 105,000 to 108,000, which is appreciably less than the lowest value (namely 118,000) to be determined by direct physico-chemical measurement. It was therefore argued (Harris, 1964) that "native" GPDH must consist of four identical protein chains which are joined together by non-covalent bonds to form an active tetramer with a molecular weight of 140,000 to 144,000. We now present evidence in support of this structure. The experiments which we describe were carried out with GPDH from pig muscle, but the over-all picture which has emerged for the structure of the pig enzyme appears to be equally valid for enzymes isolated from a variety of other species (Harris, Perham & Allison, unpublished results).
2. Methods and Results (a) Ohemicalstudies
Glyceraldehyde 3-phosphate dehydrogenase was isolated from pig muscle by the method of Elodi & Szorenyi (1956) and was recrystallized 3 times from ammonium sulphate containing redistilled ,B-mercaptoethanol (0·005 M) and EDTA (0·001 M). Enzyme activity was measured as described by Velick (1955), and the enzyme preparations used in this study were found to possess from 38,000 to 42,000 units of activity/mg of enzyme protein (expressed as the increment in optical density at 340 mJL between 15 and 45 sec X 2000, Allison & Kaplan (1964)). Enzyme preparations were stored as crystalline suspensions in ammonium sulphate (70%) at 4°C.
t Abbreviations used: GPDH, glyceraldehyde 3-phosphate dehydrogenase; S-14CM protein, 14C-labelled S-carboxymethyl protein; 14CMCys,S-[l-14C]carboxymethylcysteine; FDNB, fluorodinitrobenzene; PTH, phenylthiohydantoin.
878
J. 1. HARRIS AND R. N. PERHAM (b) Preparation of oxidized GP DH protein
The pig enzyme (80 mg; freeze-dried following dialysis against glass-distilled water) was oxidized with performic acid as described by Hirs (1956), and the oxidized protein was recovered as a freeze-dried powder. (c) Preparation of S.[l-UC]carboxymethyl protein
Pig GPDH (80 mg, estimated from its extinction coefficient at 280 mil, Dandliker & Fox (1955)) was dialysed against 0·005 M-tris-O'OOI M-EDTA at pH 7·2 for 4 hr at 2 to 4°C. To the dialysed enzyme solution (8 mgjml.) was added M-tris at pH 8·3 (0'5 mI.) followed by iodo[l-l4C]acetic acid (6mg; 32 1lmoles containing 1·1 X 10 6 cts/min/Ilmole of 14C as measured in a Nuclear-Chicago gas-flow counter). After 30 min at 0 to 4°C, solid urea was added to final concentration of 8 M; the reaction mixture was incubated at 30°C for 90 min and then dialysed at 4°C against several changes of 0·001 N-HCI. The protein derivative which precipitated initially redissolved on further dialysis, and the solution (containing 1·2 X 10 5 cts/min/mg l4C-labelled protein) was stored at-20°C. (d) Digestion with trypsin: peptide maps
(i) Oxidized GPDH
The oxidized pig enzyme (20 mg) was suspended in ammonium bicarbonate (4mI., 0'5% solution) at pH 8·0 and was digested with trypsin (1 %) at 37°C for 12 hr; the digest was completely soluble and was freeze-dried. Peptide maps were prepared as follows. A sample of the trypsin digest was dissolved in pyridine-acetic acid buffer (pH 6'5), applied to Whatman 3MM paper (1 mg digest/em), and subjected to two-dimensional ionophoresis (at 60 v/cm) in pyridine-acetic acid buffer at pH 6·5 and 3,5, respectively. The ionogram was developed with the ninhydrin reagent (1 % ninhydrin in acetone, containing 0·1 % cadmium acetate); a typical map of the trypsin digest of the oxidized pig enzyme is illustrated in Plate 1. Several duplicate ionograms were prepared and the peptide fractions which were "neutral" and "basic" at pH 6·5 could then be cut out, sewn on to fresh sheets of paper and submitted to ionophoresis at pH 3'5 and/or to chromatography in n-butanol-acetic acid-water-pyridine (30: 6: 24 : 20 by vol.). This method of treatment provided in effect a third dimension of separation for the neutral peptides: and a second dimension involving the chromatographic step proved to be superior to ionophoresis at pH 3·5 for the separation of the basic peptides. The peptide maps obtained by these methods are illustrated in Plate II. A total of 35 to 40 peptide spots was revealed with ninhydrin (and in a few instances by the o-tolidine/Kf reagent after chlorination of the peptides), of which some 8 to 10 (including free arginine) gave a positive Sakaguchi test for arginine. These observations suggested that the enzyme protein contains a total of about 35 to 40 residues of lysine plus arginine, with the possibility remaining that some of the observed peptides may represent peptide "overlaps" due to incomplete tryptic hydrolysis of poorly susceptible bonds; and peptides which are the products of the combined action of trypsin and of a chymotrypsinlike contaminant which is known to be present in the trypsin which was used for the original digestion (cf. Harris & Hindley, 1965). Little or no material was found to remain at the origin, even after chlorination and subsequent development of the peptide maps with the o-tolidine/Kf reagent. The virtual absence of an insoluble core is a noteworthy feature of the GPDH protein and means that the results of peptide mapping may be interpreted in the knowledge that the visible peptides do in fact represent most, if not all, of the amino acids which constitute the parent protein. In this respect GPDH stands in marked contrast to other proteins such as chymotrypsin (Hartley, 1964) and turnip yellow mosaic virus (Harris & Hindley, 1965), in which as much as 70% of their trypsin peptides formed insoluble cores which were not amenable to fractionation by paper methods. (ii) S-uCM-GPDH protein The S-l4CM-enzyme protein (40 mg; 0'5% solution) was digested with trypsin (1 %) in a pH-stat at pH 8'5 and 30°C for 2 hr and the soluble digest was freeze-dried.
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
879
Peptide maps were prepared by two-dimensional ionophoresis as already described, and the pattern of radioactive peptides was revealed by autoradiography (using Ilford X-ray film). As shown in Plate III, all the HC in the carboxymethylated enzyme occurs exclusively in 3 of the tryptic peptides; and although under these conditions of digestion about 10 to 20% of the digest remains at the origin, this fraction does not contain HC. The three radioactive peptides were isolated and shown to be pure by amino-acid and sequence analysis. Details of this work will be given in a separate paper, in which the isolation and characterization of all the tryptic pept.ides from the pig enzyme (Perham & Harris, unpublished results) will be described. For the present, a summary (Table I) of the results will suffice. TABLE
1
The amino acid sequence (S2T2a, in part) of UC-containing peptides formed by trypsin digestion of pig GP DH following reaction with iodo[1-uC]acetic acid in 8 M-urea S2T4a
lIe-Val-Ser-Asn -Ala-Ser-14CMCys- Thr- Thr-Asn- 14CMCys-Leu-Ala-Pro-Leu-Ala-Lys.
S2T4b
Val-Pro-Thr-Pro-Asn-Val-Ser-Val-Val-Asp-Leu- Thr- 14CMCys-Arg.
S2T2a
Gly-Tyr- Thr-Glu-Asp-Gln-Val-Ser- 14CMCys-Asp-Phe-(Asp,Asn )-Ser-Ser-Thr[His,Asxa,Thr,Ser,GlYa,Alaa,Ile2,Leu2,Phe}His-Phe-Val-Lys.
Asx, aspartyl residue where amide assignment not made.
The three radioactive peptides are shown to represent unique sequences in the primary structure of the enzyme protein. All the HC introduced into the enzyme by reaction with iodo[I-HC]acetic acid occurs as HCMCys, indicating that no other groups in the enzyme have been alkylated under the conditions chosen for the reaction. It follows therefore that the pig enzyme contains four unique residues of cysteine. Two of these occur in the peptide S2T4a, and it may be noted that its sequence, apart from the replacement of the cysteic acid residue (position II) by HCMCys, is identical with that of the oxidized "active site" peptide previously isolated from the rabbit, pig and yeast enzymes (Harris et al., 1963; Perham & Harris, 1963). This result can only mean, inter alia, that in the native enzymes the two cysteines in this peptide occur in the SH form and are not involved either in intrachain or in inter-chain disulphide bridges. Inactive enzyme preparations, on the other hand, have been shown to contain an intrachain disulphide bond between active site cysteine-7 and cysteine-Ll in each ofthe protein chains in the enzyme (Harris & Perham, 1964). (e)
Amino acid analysis
Samples of the HCM-enzyme protein were hydrolysed in sealed evacuated tubes with glass redistilled constant-boiling HCI, for 24, 48 and 72 hr, and quantitative amino acid analyses were carried out by means of a Beckmann/Spinco automatic analyser (cf. Spackman, Stein & Moore, 1958). A separate sample of the HCM-protein was digested with pepsin and was analysed for carbon-14. In this way 0·073 /Lmole HC was found to be equivalent to 0·176 /Lmole arginine, indicating a ratio of HCMCys to arginine of 4·0 to 9·8; it should be noted that this value for HCMCys is not subject to correction for destruction during acid hydrolysis. The amino-acid composition of the protein, expressed as the nearest whole numbers of individual amino acids relative to arginine as 10, is given in Table 2. These results are the mean values of several different analyses calculated after different times of hydrolysis; appropriate corrections were applied for the destruction of serine and threonine, and for the slow release of valine and isoleucine. The recovery of carboxymethylcysteine was variable; the highest value (3'5) was obtained when hydrolysis was carried out for 24 hr under high vacuum (cf, Crestfield, Moore & Stein, 1963).
880
J . 1. HARRIS AND R. N . PERHAM TABLE
2
Amino acid compositio n of glyceraldehyde 3-phosphat e dehydrogenase from pig skeletal muscle Time of hydroly sis Amino a cid
L ys H is Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp
Assumed value 24 hr]
48 hr:j:
25·8 10·6 10·0 38 ·2 21·2 16·2 19·0 13·0 33 ·6 32·6 3·9 26·3 8·9 15·7 18·0 8·6 13·5
26·9 11·1 10·0 39·2 20·6 15·4 19·2 12·4 33 ·7 32·6 29·9 8·8 18·0 18·2 8·8 13·8
Total
27 II 10 39 22& 17& 19 13 34 33 4b 30° 9 18° 18 9 14 4d 331
H ydrol ys es were carried out with 5·7 N·HCI in se a led ev ac u ated t u bes at 105°C for variou s len g th s of ti m e. The results are expressed based on an arginin e v a lue of 10·0 residues.
t Mean of 3 analyses ( ± 3 %) . :j: Mean of 2 analyse s (± 3 %). & Corrected for destructi on during 24 hr hydroly sis . The fac tors applied are se ri ne, 0 ·94; t h reon ine, 0·97 . b E stimated as cyste ic acid after p erformic oxidation or as 14CMCys after t otal ca r b oxym eth ylat ion . c Value at 48 hr and 72 hr hydrolysis. d Ca lcula t ed from the data of Velick & Furfine (1963). The chemical monomer in pig GPDH appears therefore t o consist of about 330 amino a cid residues which corresponds to a protein chain with a m olecular weight of approxim ately 36,000. The total number of lysine plus arginine residues (37) agrees well with the number of peptides (35 to 40) found by peptide mapping ofthe trypsin digest. The presence of on ly 4 residues of cy steic a cid in a comparable analysis of t he protein oxidized by performic ac id confirms that d isu lph ide bridges do not contribute t o the stru ct u re of t he nat ive en zyme. (f) E nd-group analys is
Ve lick & U de nfriend (19 53) ex a m ine d rabbit muscle and ye ast GP DH's for N -t erminal residues by means of t he FDNB m eth od, and found 1 m ole of N -terminal valine per 70, 000 g in eac h of th e t wo enzy mes. Subse q uen t ly , Halsey & N eurath (1955) report ed that approximately 2 moles of C-term ina l methionine are releas ed per mole of the yeast en zyme by the actio n of ca rboxype ptidase in the presence of 6 M-urea. T hese results su ggested that GP D H's were com po sed of two protein chains. Subsequen tl y , Deven yi, Sajgo, H orviith &
Lys Arg
+ Neutrals Glu
+ PLATB I. Peptides formed by trypsin digestion of perforrni« acid oxidized pig GPDR. Peptide map prepared by successive ionophoresis in pyridine-acetic acid at pH 6·5 and pH 3-5, 60 vJcm for 1 hr and 1-5 hr, respectively.
[facinll p. 880
+
+ Asn
Gin
Trp
, -D N P-Lys
(a)
(b)
Neutrals
Glu
Asp
+
(c)
PL ATE II. (a ) Basi" peptides fo rmed by t ryps in digestion of p erfor mi c ac id oxi di zed pig GP D H . P eptid e m ap prepared by iono phoresis in py ridine-acetic a cid, pH 6 ·5 (60 v letn for 1)0 min) a n d ch ro m atogra p hy in n -bu t a no l-ac etic a cid-wate r- py rid ine (30: 6 : 24 : 20 ). (b) N eutral p cptides formed b y trypsin d igestion of p ig GPDH oxidized by perforrnic a cid . P eptide map prepared by iono p ho res is in p yridine-ac et ic a cid (p H 3 ,5) (60 v/cm for l' li hr) and ch ro mato grap hy in n -butanol-ac eti c aci d-wa t er-pyr idi ne (30 : 6: 24 : 20) . (c) Acidic p eptides formed by t r y psin d igestion of p ig GP D H ox idi zed b y pe rform ic acid . P eptide map prepared b y succ essive io no p ho res is in p y ridin e-a ceti c a cid at pH 6·5 and pH 3 ,5, 60 v leu: for 1 hr and 2 hr, respect ively . Free glutamic a cid represents the C-te rmina l r esidue of tho protein .
S2T4b S2T40
I
S2T20
+
+ PLATE. III. R a di oaut ograph of HC-con t a ining p ept ides formed by trypsi n d igestion of P!g GPDH foll owing r ea ction with iodo[ I -14C]acetic a cid in 8 sr-urea. P eptide m ap prep a re d by successiv e ion ophor esis in p y r id ine-acetic a cid at pH 6 ·5 a nd p H 3 ·5, 60 v JCIll for 1 hr and 1·5 hr, respecti vely,
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
881
Szorenyi (1963) reported that they had obtained a yield of 1 mole of DNP-valine/ 47,000 g of enzyme protein, which supported a structure based on 3 chains. In the light of the experiments reported above, it was decided to re-investigate the end-groups in rabbit and pig muscle GPDH's. (i) FDNBmethod
Oxidized rabbit and pig enzyme proteins (40 mg) were treated with FDNB in the presence of 8 M-urea as described in Fraenkel-Conrat, Harris & Levy (1956). DPN-valine was identified as the N-terminal amino acid in both enzymes, in agreement with the results ofVelick & Udenfriend (1953) and Elodi & Szorenyi (1956). However, even after prolonged hydrolysis of the DNP-proteins (up to 24 hr at 105°C), yields of DNP-valine did not exceed I mole/55,000 g of the enzyme proteins. In addition to DNP-valine, a second yellow component (which moved close to the solvent front in the toluene solvent system) was detected; on elution and acid hydrolysis, this other component yielded additional DNPvaline together with an equivalent amount of E-DNP-lysine. Attempts to quantitate the total DNP-valine released were not altogether successful, -and the method was consequently abandoned in favour of the phenylisothiocyanate method (Edman, 1950). {iii Phenylisothiocyanate method
Analysis by this method was undertaken (using 10-mg samples of oxidized rabbit and pig enzyme proteins) by the paper strip procedure as described in Fraenkel-Conrat et al. (1956). Valine PTH was obtained from both proteins, in a yield equivalent to I mole PTH/ 38,000 g of protein. Stepwise analysis revealed further that the N -terminal sequence in the pig enzyme is Val-Lye-Val, The yield of N -terminal valine by this method is in good agreement with the results of amino-acid analysis and peptide mapping reported earlier, and it is clear that the FDNB method is giving falsely low values for the N -t erminal valine content of GPDH's. The reason for this is now apparent. The N-terminal sequence of Val-Lys-Val, particularly after dinitrophenylation, would be particularly stable to acid hydrolysis, and indeed what appears to be the peptide ot,Edi-DNP-Val-Lys (and/or ot,EdiDNP-Val-Lys-Val) has been observed intact even after prolonged hydrolysis of the DNPprotein.
3. Discussion The results of amino-acid and end-group analysis, coupled with those of peptide mapping, are consistent with the view that pig muscle GPDH is composed of identical protein chains each with a molecular weight of 36,000 ± 1000. If the true molecular weight of native active enzyme is between 140,000 and 150,000 (and the values obtained by DandIiker & Fox (1955), Fox & DandIiker (1956), E16di (1958), and Caputto & Dixon (1948, unpublished observations) are within this range), then the chemical results indicate that it must be composed of four identical chains each with a molecular weight of 36,000, rather than of three chains with a molecular weight of 40,000 to 46,000 as was suggested by the results of coenzyme binding and active site studies (cf. Velick & Furfine, 1963), and of previous end-group and fingerprint studies (Devenyi et al., 1963). The absence of disulphide bonds suggested that it should be possible to dissociate the native enzyme into its constituent chains in the presence of dissociating reagents such as sodiumdodecyl sulphate, urea, or guanidine-HCI. Preliminary studies involving sedimentation analysis in the presence of 1 % sodium dodecyl sulphate, and 8 M-urea (Perham & Harris, unpublished experiments) revealed that the anticipated dissociation into subunits oflower molecular weight does occur in the presence of these reagents. In order to obtain independent evidence for the tetrameric structure of GPDH, it became necessary to re-investigate the molecular weight of the native enzyme by
882
J. 1. HARRIS AND R. N. PERHAM
physico-chemical methods and in addition to investigate the molecular weight of the dissociated protein chains. These studies have now been carried out by W. F. Harrington and Gertrude M. Karr and the results which they have obtained are presented in the following paper (Harrington & Karr, 1965). The molecular weight of native GPDH was estimated to be 145,000 ± 6000 for the pig and rabbit muscle enzymes. In 5 M-guanidine-HCI, on the other hand, the two enzymes were found to dissociate into monodisperse subunits with a molecular weight of 36,300 ± 1500. This value for the molecular weight of the subunit in solution is in excellent agreement with the value of 36,000 ± 1000 which we have obtained for the size of the chemical monomer in the enzyme. The combined results of the chemical and physico-chemical studies indicate that the native enzyme is indeed a tetramer which is composed of four identical protein chains. These conclusions receive additional support from the results of X-ray crystallographic analysis (Watson & Banaszak, 1964), which show that the crystalline enzyme from lobster muscle possesses at least one twofold axis of symmetry. This has led them to conclude that glyceraldehyde 3-phosphate dehydrogenase, like the haemoglobins (Cullis, Muirhead, Perutz, Rossmann & North, 1962), is composed of two structurally identical halves, a result which is clearly incompatible with a structure based on three protein chains. The enzyme may therefore be pictured as an assembly of four identical protein chains, which interact with one another in a specific way so that each individual chain acquires the three-dimensional configuration (tertiary structure) which is necessary for catalytic activity within the over-all quaternary superstructure of the tetramer. It is envisaged that each active chain possesses a reactive cysteine, and that it is capable of combining with one molecule of NAD and of ortho-phosphate ion to promote the oxidation and phosphorylation of the substrate glyceraldehyde 3-phosphate. In this respect GPDH now resembles other dehydrogenases such as yeast alcohol dehydrogenase (cf. Kagi & Vallee, 1960; Harris, 1964), and the lactic dehydrogenases (Apella & Markert, 1961; Cahn, Kaplan, Levine & Zwilling, 1962), which have also been shown to be tetramers with molecular weights of approximately 150,000. Now that the tetrameric structure of pig GPDH (and of other GPDH's including rabbit, lobster and yeast enzymes (unpublished results of Harris et al.)) has been firmly established, it is of interest to reconsider the nature of previous evidence quoted in support of a trimeric structure. For example, studies of the stoicheiometry of the binding between rabbit enzyme and coenzyme indicated an equivalent combining weight of 45,000 g of enzyme protein per mole of NAD (Velick & Furfine, 1963); this result is equivalent to 2·7 moles NADj120,000 g, or of 3·2 moles NADj145,000 g of enzyme protein. Similarly, active site studies involving reaction of the mammalian enzymes with inhibitors, such as p-chloromercuribenzoate (Velick, 1953), and iodo[l- 14C]acetic acid (Harris et al., 1963), revealed the presence of 2·9 active sitesj 120,000 g of enzyme protein (equivalent to 3·5 active sitesf145,000 g). In most of these instances a value of 120,000 was assumed for the molecular weight of the enzyme and on this basis it was thought to contain three catalytic sites per mole. It should also be noted that the properties which were measured presumably apply only to active protein chains; in our experience it seems distinctly probable that any given preparation of the enzyme will not consist wholly of fully active molecules, but will contain a proportion of molecules which do not contribute to the measured
GLYCERALDEHYDE 3·PHOSPHATE DEHYDROGENASES
883
catalysis. The experimental values obtained in such measurements should therefore be related to moles of active enzyme rather than to moles of total enzyme protein. A precedent for this viewpoint is the analogous work on the inhibition of chymotrypsin by di-isopropylfluoro[32P]phosphonate (Balls & Jansen, 1952), in which the stoicheiometry of the enzyme-inhibitor reaction can be used to measure the percentage of inactive molecules present in any given preparation of the enzyme. On this basis a mole (145,000 g) ofjully active GPDH should react with 4·0 moles ofiodo[V 40]acetic acid, and p-nitrophenyl[I. 140]acetate, respectively. In the light of these considerations, there does not seem to be any fundamental disagreement between the results of the chemical and enzymological experiments. The latter appear to be in error only because they are based on a low value for the molecular weight and on the unjustified assumption that the enzyme preparations used were in all cases fully active. Yeast enzyme, in particular, is known to lose activity during isolation and on subsequent storage, even in the crystalline state (see, for example, Taylor, Meriwether and Park, 1963), and reports that this enzyme contains only two active (Velick, 1953) and NAD-binding (Stockell, 1959) sites could well be explained in this way. Just as freshly prepared crystalline preparations of rabbit enzyme have been shown to contain 3·6 moles (Racker, Klybas & Schramm, 1959) and 4·0 moles (Ferdinand, 1964; Murdock & Koeppe, 1964) of NAD per mole of enzyme, freshly prepared preparations of yeast enzyme have now been shown to contain 3·8 moles of active cysteinejl40,OOO g of enzyme protein. This result for the yeast enzyme, taken in conjunction with the results of amino-acid and end-group analysis, and of peptide mapping (Harris & Perham, unpublished results), suggests that, in common with the mammalian enzymes, yeast enzyme is also composed of four identical protein chains. All the GPDH's which have been studied (including those reported by Allison & Kaplan, 1964) have given single bands when submitted to starch gel electrophoresis, both in the native state and in the presence of 8 M-urea. There is thus no evidence for the existence of isoenzymes among glyceraldehyde 3·phosphate dehydrogenases, in contrast to the well-documented example of the lactic dehydrogenases (Appella & Markert, 1961; Cahn et al., 1962).
REFERENCES Allison, W. S. & Kaplan, N. O. (1964). J. Biol. Chem, 239, 2140. AppeIla, E. & Markert, C. L. (1961). Biochem. Biophys. Res. Comm. 6,171. Balls, A. K. & Jansen, E. F. (1952). In Advances in Enzymology, ed. by F. F. Nord, vol. 13, p. 321. New York: Interscience. Cahn, R. D., Kaplan, N. 0., Levine, L. & Zwilling, E. (1962). Science, 136, 962. Caputto, R. & Dixon, M. (1945). Nature, 156, 630. Cori, G. T., Slein, M. W. & Cori, C. F. (1945). J. Biol. Chem. 159, 565. Crestfield, A. M., Moore, S. & Stein, W. H. (1963). J. Biol. Ohern, 238, 622. CuIlis, A. F., Muirhead, H., Perutz, M. F., Rossmann, M. G. & North, A. C. T. (1962). Proc. Roy. Soc. A, 265, 161. Dandliker, W. & Fox, J. B. (1955). J. Biol. Chem, 214, 275. Devenyi, T., Sajgo, M., Horvilth, E. & Szorenyi, B. (1963). Biochim. biophys. Acta, 77,164. Edman, P. (1950). Acta Chern: Scand. 4, 277.
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Elias, R. G., Garbe, A. & Lamprecht, W. (1960). Z. physiol. Chern. 319, 22. Elodi, P. (1958). Acta Physiol. Acad. Sci. Hung. 13, 199. E16di, P. & Szorenyi, E. (1956). Acta Physiol. Acad. Sci. Hung. 9,339. Ferdinand, W. (1964). Biochem. J. 92, 578. Fox, J. B. & Dandliker, W. B. (1956). J. Biol. Chern. 218, 53. Fraenke1-Conrat, R., Harris, J. 1. & Levy, A. (1956). In Methods of Biochemical Analysis, ed. by D. Glick, vol. 2, p. 359. New York: Interscience. Halsey, Y. D. & Neurath, H. (1955). J. Biol. Chern. 217, 247. Harrington, W. F. & Karr, G. M. (1965). J. Mol. Biol. 13, 885. Harris, J. 1. (1964). In Structure and Activity of Enzymes, ed. by T. W. Goodwin, J. 1. Harris & B. S. Hartley, p. 97. London: Academic Press. Harris, J. 1. & Hindley, J. (1965). J. Mol. Biol. 13, 894. Harris, 1., Meriwether, B. P. & Park, J. H. (1963). Nature, 198, 154. Harris, J. 1. & Perham, R. N. (1963). Biochem. J. 89, 60P. Harris, J. I. & Perham, R. N. (1964). In Abstr. 6th Int. Congo Biochem. section IV, p. 293. London: Pergamon Press, in the press. Hartley, B. S. (1964). Nature, 201, 1284. Hirs, C. W. (1956). J. Biol. Chern. 219, 611. Kagi, J. H. R. & Vallee, B. L. (1960). J. Biol. Chern. 235, 3188. Murdock, A. L. & Koeppe, O. J. (1964). J. Biol. Chern. 239, 1983. Park, J. H., Meriwether, B. P., Clodfelder, P. & Cunningham, L. W. (1961). J. Biol. Chern. 236, 136. Perham, R. N. & Harris, J. 1. (1963). J. Mol. Biol. 7, 316. Racker, E., Klybas, V. & Schramm, M. (1959). J. Biol. Chern. 234, 2510. Spackman, D. H., Stein, W. H. & Moore, S. (1958). Analyt. Chern. 30, 1190. Stockell, A. (1959). J. Biol. Chern. 234, 1286. Taylor, J. F. & Lowry, S. F. (1956). Biochim, biophys. Acta, 20,109. Taylor, E. L., Meriwether, B. P. & Park, J. H. (1963). J. Biol. Chern. 238, 734. Velick, S. F. (1953). J. Biol. Chern. 203, 563. Velick, S. F. (1955). In Methods in Enzymology, ed. by S. P. Colowick & N. O. Kaplan, vol. 1, p. 401. New York: Academic Press. Velick, S. F. & Furfine, C. (1963). In The Enzymes, ed. by P. D. Boyer, H. Lardy & K. Myrback, vol. 7, p. 243. New York: Academic Press. Velick, S. F. & Udenfriend, S. (1953). J. Biol. Chern. 203, 575. Warburg, O. & Christian, W. (1939). Biochem, Z. 303, 40. Watson, H. C. & Banaszak, L. J. (1964). Nature, 204, 918.
Glyceraldehyde 3-Phosphate Dehydrogenases I. The Protein Chains in Glyceraldehyde 3-Phosphate Dehydrogenase from Pig Muscle J.
lEUAN HARRIS AND
R. N.
PERHAM
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, England (Received 4 June 1965) Glyceraldehyde 3-phosphate dehydrogenases have been studied by methods of structural protein chemistry. Derivatives of the pig muscle enzyme prepared by oxidation of the native enzyme with performic acid, and by carboxymethylation with iodo[I-14C]acetic acid in 8 M-urea, have been shown to contain four unique residues of cysteic acid, and of S-[l-14C]carboxymethylcysteine, respectively, indicating that the structural monomer in the enzyme contains four unique cysteines and that disulphide bridges do not contribute to its molecular structure. Amino-acid and end-group analysis, and a study of tryptic digests by peptide mapping techniques have shown further that the protein monomer in the pig muscle enzyme consists of a single polypeptide chain containing approximately 330 amino acid residues, corresponding to a molecular weight of 36,000 ± 1000. It is proposed that the active enzyme comprises four such identical protein chains, each of which contains one reactive cysteine and combines with one molecule of coenzyme (NAD), to form jour structurally independent catalytic sites within the quaternary structure of the active tetramer.
1. Introduction Glyceraldehyde 3-phosphate dehydrogenase (GPDH, E.C. no. 1. 2 .1.12) is a key enzyme in the glycolytic conversion of glucose to pyruvic acid and as such has an important role in the carbohydrate metabolism of most organisms (for review see Velick & Furfine, 1963). The enzyme was first obtained in pure crystalline form from yeast (Warburg & Christian, 1939) and from rabbit skeletal muscle (Caputto & Dixon, 1945; Cori, Slein & Cori, 1945). It has since been isolated from a variety of other sources, notably by Elodi & Szorenyi (1956) and Allison & Kaplan (1964). The enzyme from rabbit muscle has been variously reported to possess a molecular weight of 118,000 (Elias, Garbe & Lamprecht, 1960), 120,000 (Taylor & Lowry, 1956), 140,000 (Dandliker & Fox, 1955; Fox & Dandliker, 1956) and 142,000 (EMdi, 1958), and this apparent variability in molecular weight is reflected in the values reported for other parameters of the active enzyme, such as the number of moles of bound coenzyme (NAD) and the number of catalytically active sites. A molecular weight of 120,000 has been frequently adopted; on this basis the rabbit enzyme was thought to combine with 3 moles of NAD, and to contain from 11 to 14 titratable SH groups, of which 3 appeared to be highly reactive and essential for activity (cf. Velick & Furfine, 1963). 876
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
877
The determination of the amino acid sequences around the reactive cysteines in GPDH'st following their reaction with the inhibitor, iodo[l- 14C]acetic acid, and the substrate, p-nitrophenyl[l- 14C]acetate, respectively, showed that these particular cysteines occurred in the same octadecapeptide sequence in the primary structure of the enzyme protein. These results led us to suggest that a molecule of active enzyme contained at least three catalytic sites and that it was composed of an equal number of similar, if not identical, protein chains (Harris, Meriwether & Park, 1963; Harris & Perham, 1963). At the same time it was apparent that this estimate of the number of constituent protein chains represented a minimum value, which was based on the assumption that the enzyme preparations used for these studies were composed wholly of fully active enzyme molecules. If, on the other hand, they contained a proportion of protein chains which were not capable of reacting with the inhibitor or the substrate, then the above estimate of the number of protein chains comprising the enzyme molecule could well be too low. In an endeavour to eliminate this possible source of error, we have sought to establish the number, and molecular size, of the protein chains in glyceraldehyde 3-phosphate dehydrogenases by methods which do not necessarily depend on the availability offully active enzyme. A study of the enzyme protein, involving amino-acid and endgroup analysis and the characterization of polypeptide fragments produced by trypsin digestion of the carboxymethylated derivatives of rabbit and pig muscle GPDH's, has now revealed that the structural monomer in both enzymes appears to consist of a protein chain containing approximately 330 amino acids, corresponding to a chain molecular weight of 35,000 to 36,000 (Harris & Perham, 1963,1964). On this basis, active enzyme consisting of three of these protein chains would be expected to possess a molecular weight of 105,000 to 108,000, which is appreciably less than the lowest value (namely 118,000) to be determined by direct physico-chemical measurement. It was therefore argued (Harris, 1964) that "native" GPDH must consist of four identical protein chains which are joined together by non-covalent bonds to form an active tetramer with a molecular weight of 140,000 to 144,000. We now present evidence in support of this structure. The experiments which we describe were carried out with GPDH from pig muscle, but the over-all picture which has emerged for the structure of the pig enzyme appears to be equally valid for enzymes isolated from a variety of other species (Harris, Perham & Allison, unpublished results).
2. Methods and Results (a) Ohemicalstudies
Glyceraldehyde 3-phosphate dehydrogenase was isolated from pig muscle by the method of Elodi & Szorenyi (1956) and was recrystallized 3 times from ammonium sulphate containing redistilled ,B-mercaptoethanol (0·005 M) and EDTA (0·001 M). Enzyme activity was measured as described by Velick (1955), and the enzyme preparations used in this study were found to possess from 38,000 to 42,000 units of activity/mg of enzyme protein (expressed as the increment in optical density at 340 mJL between 15 and 45 sec X 2000, Allison & Kaplan (1964)). Enzyme preparations were stored as crystalline suspensions in ammonium sulphate (70%) at 4°C.
t Abbreviations used: GPDH, glyceraldehyde 3-phosphate dehydrogenase; S-14CM protein, 14C-labelled S-carboxymethyl protein; 14CMCys,S-[l-14C]carboxymethylcysteine; FDNB, fluorodinitrobenzene; PTH, phenylthiohydantoin.
878
J. 1. HARRIS AND R. N. PERHAM (b) Preparation of oxidized GP DH protein
The pig enzyme (80 mg; freeze-dried following dialysis against glass-distilled water) was oxidized with performic acid as described by Hirs (1956), and the oxidized protein was recovered as a freeze-dried powder. (c) Preparation of S.[l-UC]carboxymethyl protein
Pig GPDH (80 mg, estimated from its extinction coefficient at 280 mil, Dandliker & Fox (1955)) was dialysed against 0·005 M-tris-O'OOI M-EDTA at pH 7·2 for 4 hr at 2 to 4°C. To the dialysed enzyme solution (8 mgjml.) was added M-tris at pH 8·3 (0'5 mI.) followed by iodo[l-l4C]acetic acid (6mg; 32 1lmoles containing 1·1 X 10 6 cts/min/Ilmole of 14C as measured in a Nuclear-Chicago gas-flow counter). After 30 min at 0 to 4°C, solid urea was added to final concentration of 8 M; the reaction mixture was incubated at 30°C for 90 min and then dialysed at 4°C against several changes of 0·001 N-HCI. The protein derivative which precipitated initially redissolved on further dialysis, and the solution (containing 1·2 X 10 5 cts/min/mg l4C-labelled protein) was stored at-20°C. (d) Digestion with trypsin: peptide maps
(i) Oxidized GPDH
The oxidized pig enzyme (20 mg) was suspended in ammonium bicarbonate (4mI., 0'5% solution) at pH 8·0 and was digested with trypsin (1 %) at 37°C for 12 hr; the digest was completely soluble and was freeze-dried. Peptide maps were prepared as follows. A sample of the trypsin digest was dissolved in pyridine-acetic acid buffer (pH 6'5), applied to Whatman 3MM paper (1 mg digest/em), and subjected to two-dimensional ionophoresis (at 60 v/cm) in pyridine-acetic acid buffer at pH 6·5 and 3,5, respectively. The ionogram was developed with the ninhydrin reagent (1 % ninhydrin in acetone, containing 0·1 % cadmium acetate); a typical map of the trypsin digest of the oxidized pig enzyme is illustrated in Plate 1. Several duplicate ionograms were prepared and the peptide fractions which were "neutral" and "basic" at pH 6·5 could then be cut out, sewn on to fresh sheets of paper and submitted to ionophoresis at pH 3'5 and/or to chromatography in n-butanol-acetic acid-water-pyridine (30: 6: 24 : 20 by vol.). This method of treatment provided in effect a third dimension of separation for the neutral peptides: and a second dimension involving the chromatographic step proved to be superior to ionophoresis at pH 3·5 for the separation of the basic peptides. The peptide maps obtained by these methods are illustrated in Plate II. A total of 35 to 40 peptide spots was revealed with ninhydrin (and in a few instances by the o-tolidine/Kf reagent after chlorination of the peptides), of which some 8 to 10 (including free arginine) gave a positive Sakaguchi test for arginine. These observations suggested that the enzyme protein contains a total of about 35 to 40 residues of lysine plus arginine, with the possibility remaining that some of the observed peptides may represent peptide "overlaps" due to incomplete tryptic hydrolysis of poorly susceptible bonds; and peptides which are the products of the combined action of trypsin and of a chymotrypsinlike contaminant which is known to be present in the trypsin which was used for the original digestion (cf. Harris & Hindley, 1965). Little or no material was found to remain at the origin, even after chlorination and subsequent development of the peptide maps with the o-tolidine/Kf reagent. The virtual absence of an insoluble core is a noteworthy feature of the GPDH protein and means that the results of peptide mapping may be interpreted in the knowledge that the visible peptides do in fact represent most, if not all, of the amino acids which constitute the parent protein. In this respect GPDH stands in marked contrast to other proteins such as chymotrypsin (Hartley, 1964) and turnip yellow mosaic virus (Harris & Hindley, 1965), in which as much as 70% of their trypsin peptides formed insoluble cores which were not amenable to fractionation by paper methods. (ii) S-uCM-GPDH protein The S-l4CM-enzyme protein (40 mg; 0'5% solution) was digested with trypsin (1 %) in a pH-stat at pH 8'5 and 30°C for 2 hr and the soluble digest was freeze-dried.
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
879
Peptide maps were prepared by two-dimensional ionophoresis as already described, and the pattern of radioactive peptides was revealed by autoradiography (using Ilford X-ray film). As shown in Plate III, all the HC in the carboxymethylated enzyme occurs exclusively in 3 of the tryptic peptides; and although under these conditions of digestion about 10 to 20% of the digest remains at the origin, this fraction does not contain HC. The three radioactive peptides were isolated and shown to be pure by amino-acid and sequence analysis. Details of this work will be given in a separate paper, in which the isolation and characterization of all the tryptic pept.ides from the pig enzyme (Perham & Harris, unpublished results) will be described. For the present, a summary (Table I) of the results will suffice. TABLE
1
The amino acid sequence (S2T2a, in part) of UC-containing peptides formed by trypsin digestion of pig GP DH following reaction with iodo[1-uC]acetic acid in 8 M-urea S2T4a
lIe-Val-Ser-Asn -Ala-Ser-14CMCys- Thr- Thr-Asn- 14CMCys-Leu-Ala-Pro-Leu-Ala-Lys.
S2T4b
Val-Pro-Thr-Pro-Asn-Val-Ser-Val-Val-Asp-Leu- Thr- 14CMCys-Arg.
S2T2a
Gly-Tyr- Thr-Glu-Asp-Gln-Val-Ser- 14CMCys-Asp-Phe-(Asp,Asn )-Ser-Ser-Thr[His,Asxa,Thr,Ser,GlYa,Alaa,Ile2,Leu2,Phe}His-Phe-Val-Lys.
Asx, aspartyl residue where amide assignment not made.
The three radioactive peptides are shown to represent unique sequences in the primary structure of the enzyme protein. All the HC introduced into the enzyme by reaction with iodo[I-HC]acetic acid occurs as HCMCys, indicating that no other groups in the enzyme have been alkylated under the conditions chosen for the reaction. It follows therefore that the pig enzyme contains four unique residues of cysteine. Two of these occur in the peptide S2T4a, and it may be noted that its sequence, apart from the replacement of the cysteic acid residue (position II) by HCMCys, is identical with that of the oxidized "active site" peptide previously isolated from the rabbit, pig and yeast enzymes (Harris et al., 1963; Perham & Harris, 1963). This result can only mean, inter alia, that in the native enzymes the two cysteines in this peptide occur in the SH form and are not involved either in intrachain or in inter-chain disulphide bridges. Inactive enzyme preparations, on the other hand, have been shown to contain an intrachain disulphide bond between active site cysteine-7 and cysteine-Ll in each ofthe protein chains in the enzyme (Harris & Perham, 1964). (e)
Amino acid analysis
Samples of the HCM-enzyme protein were hydrolysed in sealed evacuated tubes with glass redistilled constant-boiling HCI, for 24, 48 and 72 hr, and quantitative amino acid analyses were carried out by means of a Beckmann/Spinco automatic analyser (cf. Spackman, Stein & Moore, 1958). A separate sample of the HCM-protein was digested with pepsin and was analysed for carbon-14. In this way 0·073 /Lmole HC was found to be equivalent to 0·176 /Lmole arginine, indicating a ratio of HCMCys to arginine of 4·0 to 9·8; it should be noted that this value for HCMCys is not subject to correction for destruction during acid hydrolysis. The amino-acid composition of the protein, expressed as the nearest whole numbers of individual amino acids relative to arginine as 10, is given in Table 2. These results are the mean values of several different analyses calculated after different times of hydrolysis; appropriate corrections were applied for the destruction of serine and threonine, and for the slow release of valine and isoleucine. The recovery of carboxymethylcysteine was variable; the highest value (3'5) was obtained when hydrolysis was carried out for 24 hr under high vacuum (cf, Crestfield, Moore & Stein, 1963).
880
J . 1. HARRIS AND R. N . PERHAM TABLE
2
Amino acid compositio n of glyceraldehyde 3-phosphat e dehydrogenase from pig skeletal muscle Time of hydroly sis Amino a cid
L ys H is Arg Asp Thr Ser Glu Pro Gly Ala Cys Val Met lie Leu Tyr Phe Trp
Assumed value 24 hr]
48 hr:j:
25·8 10·6 10·0 38 ·2 21·2 16·2 19·0 13·0 33 ·6 32·6 3·9 26·3 8·9 15·7 18·0 8·6 13·5
26·9 11·1 10·0 39·2 20·6 15·4 19·2 12·4 33 ·7 32·6 29·9 8·8 18·0 18·2 8·8 13·8
Total
27 II 10 39 22& 17& 19 13 34 33 4b 30° 9 18° 18 9 14 4d 331
H ydrol ys es were carried out with 5·7 N·HCI in se a led ev ac u ated t u bes at 105°C for variou s len g th s of ti m e. The results are expressed based on an arginin e v a lue of 10·0 residues.
t Mean of 3 analyses ( ± 3 %) . :j: Mean of 2 analyse s (± 3 %). & Corrected for destructi on during 24 hr hydroly sis . The fac tors applied are se ri ne, 0 ·94; t h reon ine, 0·97 . b E stimated as cyste ic acid after p erformic oxidation or as 14CMCys after t otal ca r b oxym eth ylat ion . c Value at 48 hr and 72 hr hydrolysis. d Ca lcula t ed from the data of Velick & Furfine (1963). The chemical monomer in pig GPDH appears therefore t o consist of about 330 amino a cid residues which corresponds to a protein chain with a m olecular weight of approxim ately 36,000. The total number of lysine plus arginine residues (37) agrees well with the number of peptides (35 to 40) found by peptide mapping ofthe trypsin digest. The presence of on ly 4 residues of cy steic a cid in a comparable analysis of t he protein oxidized by performic ac id confirms that d isu lph ide bridges do not contribute t o the stru ct u re of t he nat ive en zyme. (f) E nd-group analys is
Ve lick & U de nfriend (19 53) ex a m ine d rabbit muscle and ye ast GP DH's for N -t erminal residues by means of t he FDNB m eth od, and found 1 m ole of N -terminal valine per 70, 000 g in eac h of th e t wo enzy mes. Subse q uen t ly , Halsey & N eurath (1955) report ed that approximately 2 moles of C-term ina l methionine are releas ed per mole of the yeast en zyme by the actio n of ca rboxype ptidase in the presence of 6 M-urea. T hese results su ggested that GP D H's were com po sed of two protein chains. Subsequen tl y , Deven yi, Sajgo, H orviith &
Lys Arg
+ Neutrals Glu
+ PLATB I. Peptides formed by trypsin digestion of perforrni« acid oxidized pig GPDR. Peptide map prepared by successive ionophoresis in pyridine-acetic acid at pH 6·5 and pH 3-5, 60 vJcm for 1 hr and 1-5 hr, respectively.
[facinll p. 880
+
+ Asn
Gin
Trp
, -D N P-Lys
(a)
(b)
Neutrals
Glu
Asp
+
(c)
PL ATE II. (a ) Basi" peptides fo rmed by t ryps in digestion of p erfor mi c ac id oxi di zed pig GP D H . P eptid e m ap prepared by iono phoresis in py ridine-acetic a cid, pH 6 ·5 (60 v letn for 1)0 min) a n d ch ro m atogra p hy in n -bu t a no l-ac etic a cid-wate r- py rid ine (30: 6 : 24 : 20 ). (b) N eutral p cptides formed b y trypsin d igestion of p ig GPDH oxidized by perforrnic a cid . P eptide map prepared by iono p ho res is in p yridine-ac et ic a cid (p H 3 ,5) (60 v/cm for l' li hr) and ch ro mato grap hy in n -butanol-ac eti c aci d-wa t er-pyr idi ne (30 : 6: 24 : 20) . (c) Acidic p eptides formed by t r y psin d igestion of p ig GP D H ox idi zed b y pe rform ic acid . P eptide map prepared b y succ essive io no p ho res is in p y ridin e-a ceti c a cid at pH 6·5 and pH 3 ,5, 60 v leu: for 1 hr and 2 hr, respect ively . Free glutamic a cid represents the C-te rmina l r esidue of tho protein .
S2T4b S2T40
I
S2T20
+
+ PLATE. III. R a di oaut ograph of HC-con t a ining p ept ides formed by trypsi n d igestion of P!g GPDH foll owing r ea ction with iodo[ I -14C]acetic a cid in 8 sr-urea. P eptide m ap prep a re d by successiv e ion ophor esis in p y r id ine-acetic a cid at pH 6 ·5 a nd p H 3 ·5, 60 v JCIll for 1 hr and 1·5 hr, respecti vely,
GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASES
881
Szorenyi (1963) reported that they had obtained a yield of 1 mole of DNP-valine/ 47,000 g of enzyme protein, which supported a structure based on 3 chains. In the light of the experiments reported above, it was decided to re-investigate the end-groups in rabbit and pig muscle GPDH's. (i) FDNBmethod
Oxidized rabbit and pig enzyme proteins (40 mg) were treated with FDNB in the presence of 8 M-urea as described in Fraenkel-Conrat, Harris & Levy (1956). DPN-valine was identified as the N-terminal amino acid in both enzymes, in agreement with the results ofVelick & Udenfriend (1953) and Elodi & Szorenyi (1956). However, even after prolonged hydrolysis of the DNP-proteins (up to 24 hr at 105°C), yields of DNP-valine did not exceed I mole/55,000 g of the enzyme proteins. In addition to DNP-valine, a second yellow component (which moved close to the solvent front in the toluene solvent system) was detected; on elution and acid hydrolysis, this other component yielded additional DNPvaline together with an equivalent amount of E-DNP-lysine. Attempts to quantitate the total DNP-valine released were not altogether successful, -and the method was consequently abandoned in favour of the phenylisothiocyanate method (Edman, 1950). {iii Phenylisothiocyanate method
Analysis by this method was undertaken (using 10-mg samples of oxidized rabbit and pig enzyme proteins) by the paper strip procedure as described in Fraenkel-Conrat et al. (1956). Valine PTH was obtained from both proteins, in a yield equivalent to I mole PTH/ 38,000 g of protein. Stepwise analysis revealed further that the N -terminal sequence in the pig enzyme is Val-Lye-Val, The yield of N -terminal valine by this method is in good agreement with the results of amino-acid analysis and peptide mapping reported earlier, and it is clear that the FDNB method is giving falsely low values for the N -t erminal valine content of GPDH's. The reason for this is now apparent. The N-terminal sequence of Val-Lys-Val, particularly after dinitrophenylation, would be particularly stable to acid hydrolysis, and indeed what appears to be the peptide ot,Edi-DNP-Val-Lys (and/or ot,EdiDNP-Val-Lys-Val) has been observed intact even after prolonged hydrolysis of the DNPprotein.
3. Discussion The results of amino-acid and end-group analysis, coupled with those of peptide mapping, are consistent with the view that pig muscle GPDH is composed of identical protein chains each with a molecular weight of 36,000 ± 1000. If the true molecular weight of native active enzyme is between 140,000 and 150,000 (and the values obtained by DandIiker & Fox (1955), Fox & DandIiker (1956), E16di (1958), and Caputto & Dixon (1948, unpublished observations) are within this range), then the chemical results indicate that it must be composed of four identical chains each with a molecular weight of 36,000, rather than of three chains with a molecular weight of 40,000 to 46,000 as was suggested by the results of coenzyme binding and active site studies (cf. Velick & Furfine, 1963), and of previous end-group and fingerprint studies (Devenyi et al., 1963). The absence of disulphide bonds suggested that it should be possible to dissociate the native enzyme into its constituent chains in the presence of dissociating reagents such as sodiumdodecyl sulphate, urea, or guanidine-HCI. Preliminary studies involving sedimentation analysis in the presence of 1 % sodium dodecyl sulphate, and 8 M-urea (Perham & Harris, unpublished experiments) revealed that the anticipated dissociation into subunits oflower molecular weight does occur in the presence of these reagents. In order to obtain independent evidence for the tetrameric structure of GPDH, it became necessary to re-investigate the molecular weight of the native enzyme by
882
J. 1. HARRIS AND R. N. PERHAM
physico-chemical methods and in addition to investigate the molecular weight of the dissociated protein chains. These studies have now been carried out by W. F. Harrington and Gertrude M. Karr and the results which they have obtained are presented in the following paper (Harrington & Karr, 1965). The molecular weight of native GPDH was estimated to be 145,000 ± 6000 for the pig and rabbit muscle enzymes. In 5 M-guanidine-HCI, on the other hand, the two enzymes were found to dissociate into monodisperse subunits with a molecular weight of 36,300 ± 1500. This value for the molecular weight of the subunit in solution is in excellent agreement with the value of 36,000 ± 1000 which we have obtained for the size of the chemical monomer in the enzyme. The combined results of the chemical and physico-chemical studies indicate that the native enzyme is indeed a tetramer which is composed of four identical protein chains. These conclusions receive additional support from the results of X-ray crystallographic analysis (Watson & Banaszak, 1964), which show that the crystalline enzyme from lobster muscle possesses at least one twofold axis of symmetry. This has led them to conclude that glyceraldehyde 3-phosphate dehydrogenase, like the haemoglobins (Cullis, Muirhead, Perutz, Rossmann & North, 1962), is composed of two structurally identical halves, a result which is clearly incompatible with a structure based on three protein chains. The enzyme may therefore be pictured as an assembly of four identical protein chains, which interact with one another in a specific way so that each individual chain acquires the three-dimensional configuration (tertiary structure) which is necessary for catalytic activity within the over-all quaternary superstructure of the tetramer. It is envisaged that each active chain possesses a reactive cysteine, and that it is capable of combining with one molecule of NAD and of ortho-phosphate ion to promote the oxidation and phosphorylation of the substrate glyceraldehyde 3-phosphate. In this respect GPDH now resembles other dehydrogenases such as yeast alcohol dehydrogenase (cf. Kagi & Vallee, 1960; Harris, 1964), and the lactic dehydrogenases (Apella & Markert, 1961; Cahn, Kaplan, Levine & Zwilling, 1962), which have also been shown to be tetramers with molecular weights of approximately 150,000. Now that the tetrameric structure of pig GPDH (and of other GPDH's including rabbit, lobster and yeast enzymes (unpublished results of Harris et al.)) has been firmly established, it is of interest to reconsider the nature of previous evidence quoted in support of a trimeric structure. For example, studies of the stoicheiometry of the binding between rabbit enzyme and coenzyme indicated an equivalent combining weight of 45,000 g of enzyme protein per mole of NAD (Velick & Furfine, 1963); this result is equivalent to 2·7 moles NADj120,000 g, or of 3·2 moles NADj145,000 g of enzyme protein. Similarly, active site studies involving reaction of the mammalian enzymes with inhibitors, such as p-chloromercuribenzoate (Velick, 1953), and iodo[l- 14C]acetic acid (Harris et al., 1963), revealed the presence of 2·9 active sitesj 120,000 g of enzyme protein (equivalent to 3·5 active sitesf145,000 g). In most of these instances a value of 120,000 was assumed for the molecular weight of the enzyme and on this basis it was thought to contain three catalytic sites per mole. It should also be noted that the properties which were measured presumably apply only to active protein chains; in our experience it seems distinctly probable that any given preparation of the enzyme will not consist wholly of fully active molecules, but will contain a proportion of molecules which do not contribute to the measured
GLYCERALDEHYDE 3·PHOSPHATE DEHYDROGENASES
883
catalysis. The experimental values obtained in such measurements should therefore be related to moles of active enzyme rather than to moles of total enzyme protein. A precedent for this viewpoint is the analogous work on the inhibition of chymotrypsin by di-isopropylfluoro[32P]phosphonate (Balls & Jansen, 1952), in which the stoicheiometry of the enzyme-inhibitor reaction can be used to measure the percentage of inactive molecules present in any given preparation of the enzyme. On this basis a mole (145,000 g) ofjully active GPDH should react with 4·0 moles ofiodo[V 40]acetic acid, and p-nitrophenyl[I. 140]acetate, respectively. In the light of these considerations, there does not seem to be any fundamental disagreement between the results of the chemical and enzymological experiments. The latter appear to be in error only because they are based on a low value for the molecular weight and on the unjustified assumption that the enzyme preparations used were in all cases fully active. Yeast enzyme, in particular, is known to lose activity during isolation and on subsequent storage, even in the crystalline state (see, for example, Taylor, Meriwether and Park, 1963), and reports that this enzyme contains only two active (Velick, 1953) and NAD-binding (Stockell, 1959) sites could well be explained in this way. Just as freshly prepared crystalline preparations of rabbit enzyme have been shown to contain 3·6 moles (Racker, Klybas & Schramm, 1959) and 4·0 moles (Ferdinand, 1964; Murdock & Koeppe, 1964) of NAD per mole of enzyme, freshly prepared preparations of yeast enzyme have now been shown to contain 3·8 moles of active cysteinejl40,OOO g of enzyme protein. This result for the yeast enzyme, taken in conjunction with the results of amino-acid and end-group analysis, and of peptide mapping (Harris & Perham, unpublished results), suggests that, in common with the mammalian enzymes, yeast enzyme is also composed of four identical protein chains. All the GPDH's which have been studied (including those reported by Allison & Kaplan, 1964) have given single bands when submitted to starch gel electrophoresis, both in the native state and in the presence of 8 M-urea. There is thus no evidence for the existence of isoenzymes among glyceraldehyde 3·phosphate dehydrogenases, in contrast to the well-documented example of the lactic dehydrogenases (Appella & Markert, 1961; Cahn et al., 1962).
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